Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA. Genes for the latter two enzymes, tatA and batA, were previously isolated from R. meliloti. aatB is distinct from but hybridizes to aatA, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da. The amino acid sequence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs. AatB expressed in E. coli has a Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed in aatB and tatA and transferred to the genome of R. meliloti 104A14. Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation.